Search

Striatin and SG2NA are scaffold proteins that form signaling complexes called STRIPAK. It has been associated with developmental abnormalities, cancer, and several other diseases. Our earlier studies have shown that SG2NA forms a complex with the cancer-associated protein DJ-1 and the signaling kinase Akt, promoting cancer cell survival. In the present study, we used bioinformatics analyses to confirm the existence of two isoforms of human SG2NA, i.e., 78 and 87 kDas. In addition, several smaller isoforms like 35 kDa were also seen in western blot analyses of human cell lysates. The expression of these isoforms varies between different cancer cell lines of human origin. Also, the protein levels do not corroborate with its transcript levels, suggesting a complex regulation of its expression. In breast tumor tissues, the expression of the 35 and 78 kDa isoforms was higher as compared to the adjacent normal tissues, while the 87 kDa isoform was found in the breast tumor tissues only. With the progression of stages of breast cancer, while the expression of 78 kDa isoform decreased, 87 kDa became undetectable. In co-immunoprecipitation assays, the profile of the SG2NA interactome in breast tumors vis-à-vis adjacent normal breast tissues showed hundreds of common proteins. Also, some proteins were interacted with SG2NA in breast tumor tissues only. We conclude that SG2NA is involved in diverse cellular pathways and has roles in cellular reprogramming during tumorigenesis of the breast.

SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffold for multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)-tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3β-ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP-tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP-tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation-dephosphorylation. The inhibition of GSK3β by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP-tagged 35, 52, and 87 kDa, inhibition of GSK3β caused an initial increase followed by a decrease with a subtle difference in kinetics and intensities. Similar results were also seen upon inhibition of GSK3β by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibition of extracellular-signal-regulated-kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3β, and ERK. Taken together, the variants of SG2NA are modulated by the kinase-phosphatase network in a similar but characteristic manner.

SG2NA is a protein of the striatin family that organizes STRIPAK complexes. It has splice variants expressing differentially in tissues. Its 78 kDa isoform regulates cell cycle, maintains homeostasis in the endoplasmic reticulum, and prevents oxidative injuries. The 35 kDa variant is devoid of the signature WD-40 repeats in the carboxy terminal, and its function is unknown. We expressed it in NIH 3T3 cells that otherwise express 78 kDa variant only. These cells (35 EE) have altered morphology, faster rate of migration, and enhanced growth as measured by the MTT assay. Similar phenotypes were also seen in cells where the endogenous 78 kDa isoform was downregulated by siRNA (78 KD). Proteomic analyses showed that several cancer-associated proteins are modulated in both 35 EE and 78 KD cells. The 35 EE cells have diffused actin fibers, distinctive ultrastructure, reduced sialylation, and increased expression of MMP2 & 9. The 78 KD cells also had diffused actin fibers and an upregulated expression of MMP2. In both cells, markers epithelial to mesenchymal transition (EMT) viz, E- & N-cadherins, β-catenin, slug, vimentin, and ZO-1 were modulated partially in tune with the EMT process. Since NIH 3T3 cells are mesenchymal, we also expressed 35 kDa SG2NA in MCF-7 cells of epithelial origin. In these cells (MCF-7-35), the actin fibers were also diffused and the modulation of the markers was more in tune with the EMT process. However, unlike in 35 EE cells, in MCF-7-35 cells, membrane sialylation rather increased. We infer that ectopic expression of 35 kDa and downregulation of 78 kDa SG2NAs partially induce transformed phenotypes.

The WD40 domain is one of the most occurring domains in the eukaryotic genome. Recently it is also reported in prokaryotes. The protein with WD40 domain acts as scaffold proteins and helps in the assembly of multiprotein complexes. Genome wide identification and analysis of WD40 domain-containing proteins in Zebrafish was performed in the present work. Total 251 redundant proteins with WD40 domain (at least six WD40 repeats) were identified from WDSP software and further confirmed using ZFIN and NCBI database. Their chromosomal localization and domain architecture was analyzed. The DrWD40 genes are distributed on all the chromosomes with the maximum number (18) on chromosome 5. Based on the presence of other domains, DrWD40 proteins were classified into 18 classes. The coiled-coil domain was the most occurring domain along with the WD40 domain. In the KEGG pathway mapping, they show involvement in different pathways viz., RNA transport, mRNA surveillance, ribosome biogenesis, etc. In gene ontology analysis using DAVID and PANTHER software, it was found that they are involved in diverse biological processes and perform different molecular functions. WD40 proteins in Danio rerio predominantly participate in a binding activity such as actin filament binding, microtubule-binding, dynein binding, etc. They are mainly localized to the cytoplasm and nucleus. Our analysis highlighted the participation of DrWD40 proteins in diverse cellular and biological processes. A cluster of proteins with similar molecular and biological functions can be further studied.

Regulation of gene expression is attributed at multiple levels as transcriptional, post-transcriptional and post-translational events. Under various modes of post-transcriptional regulation, alternative polyadenylation (APA) is one of the means to generate transcriptome diversity. In alternative polyadenylation, multiple transcripts are generated from a single gene by the use of the different polyadenylation site in the 3′UTR and other regions. It is a highly regulated process as deregulation in this is associated with the pathogenesis of various diseases. Cancer-associated genes can avoid the miRNA mediated negative regulation by using the proximal polyadenylation signal (PAS) and shorten the length of 3′UTR. Highly proliferative or cancer cells express the transcripts with short 3′UTR which lacks binding sites for many miRNA and regulatory RNA binding proteins (RBPs). Shortening of the 3′UTR associated with tumor aggressiveness and poor survival of cancer patients. Global analysis of APA is now a focused area of study in context to cancer progression. In this review, we will comprehensively discuss the alternative polyadenylation process and their implication in cancer.

SG2NA was first discovered as nuclear autoantigen in lung and bladder cancer patient. It was named SG2NA as its expression increases during S to G2 phase of cell cycle. SG2NA/Striatin3 was classified as a member of Striatin family along with Straitin and Zinedin due to its structural and functional relatedness. At the molecular level, SG2NA is characterized by the presence of multiple protein–protein interaction domains viz., a caveolin binding motif, a coiled coil structure, Ca2+—calmodulin binding domain and a large WD-40 repeat domain in the same order from amino to the carboxyl termini. Analysis of secondary structures of 87 and 78 kDa SG2NA isoforms showed characteristic combinations of α-helix, β-structure, β-turns and random coil; suggesting of effective refolding after denaturation. This study for the first time establishes the structural differences between the two prevalent isoforms of SG2NA. Recently we observed that DJ-1 interacts with variants of SG2NA both in vitro and in vivo. The SG2NA isoforms purified from inclusion bodies showed the different secondary structure conformations, stability and interaction pattern for their interacting partners (DJ-1 and calmodulin) which imparts functional diversity of SG2NA. The SG2NA isoforms showed significant differential binding affinity to DJ-1 and Calmodulin.

The WD40 domain is one of the most abundant and interacting domains in the eukaryotic genome. In proteins the WD domain folds into a β-propeller structure, providing a platform for the interaction and assembly of several proteins into a signalosome. WD40 repeats containing proteins, in lower eukaryotes, are mainly involved in growth, cell cycle, development and virulence, while in higher organisms, they play an important role in diverse cellular functions like signal transduction, cell cycle control, intracellular transport, chromatin remodelling, cytoskeletal organization, apoptosis, development, transcriptional regulation, immune responses. To play the regulatory role in various processes, they act as a scaffold for protein-protein or protein-DNA interaction. So far, no WD40 domain has been identified with intrinsic enzymatic activity. Several WD40 domain-containing proteins have been recently characterized in prokaryotes as well. The review summarizes the vast array of functions performed by different WD40 domain containing proteins, their domain organization and functional conservation during the course of evolution.

Striatin and SG2NA are essential constituents of the multi-protein STRIPAK assembly harbouring protein phosphatase PP2A and several kinases. SG2NA has several isoforms generated by mRNA splicing and editing. While the expression of striatin is largely restricted to the striatum in brain, that of SG2NAs is ubiquitous. In NIH3T3 cells, only the 78 kDa isoform is expressed. When cells enter into the S phase, the level of SG2NA increases; reaches maximum at the G2/M phase and declines thereafter. Downregulation of SG2NA extends G1 phase and its overexpression extends G2. Ectopic expression of the 35 kDa has no effects on the cell cycle. Relative abundance of phospho-SG2NA is high in the microsome and cytosol and the nucleus but low in the mitochondria. Okadoic acid, an inhibitor of PP2A, increases the level of SG2NA which is further enhanced upon inhibition of proteasomal activity. Phospho-SG2NA is thus more stable than the dephosphorylated form. Inhibition of GSK3β by LiCl reduces its level, but the inhibition of ERK by PD98059 increases it. Thus, ERK decreases the level of phospho-SG2NA by inhibiting GSK3β. In cells depleted from SG2NA by shRNA, the levels of pGSK3β and pERK are reduced, suggesting that these kinases and SG2NA regulate each other’s expression.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The WD40 domain containing proteins are present in the lower organisms (Monera) to higher complex metazoans with involvement in diverse cellular processes. The WD40 repeats fold into β propeller structure due to which the proteins harbouring WD40 domains function as scaffold by offering platform for interactions, bring together diverse cellular proteins to form a single complex for mediating downstream effects. Multiple functions of WD40 domain containing proteins in lower eukaryote as in Fungi have been reported with involvement in vegetative and reproductive growth, virulence etc. In this article insilico analysis of the WDR proteins in the budding yeast Saccharomyces cerevisiae was performed. By WDSP software 83 proteins in S. cerevisiae were identified with at least one WD40 motif. WD40 proteins with 6 or more WD40 motifs were considered for further studies. The WD40 proteins in yeast which are involved in various biological processes show distribution on all chromosomes (16 chromosomes in yeast) except chromosome 1. Besides the WD40 domain some of these proteins also contain other protein domains which might be responsible for the diversity in the functions of WD40 proteins in the budding yeast. These proteins in budding yeast were analysed by DAVID and Blast2Go software for functional and domains categorization. Candida albicans, an opportunistic fungal pathogen also have orthologs of these WD40 proteins with possible similar functions. This is the first time genome wide analysis of WD40 proteins in lower eukaryote i.e. budding yeast. This data may be useful in further study of the functional diversity of yeast proteomes.

SG2NA belongs to a three member Striatin subfamily of WD-40 repeat superfamily. It has multiple protein-protein interaction domains that are involved in the assembly of supra-molecular signaling complexes. Earlier we had demonstrated that there are at least five variants of SG2NA, generated by alternative splicing. We now demonstrate that a 52kDa novel variant is generated by the editing of the transcript for the 82kDa isoform. The 52kDa protein is abundant in mouse tissues but it is barely present in immortalized cells, suggesting its role in cell differentiation. Besides splicing and editing, expression of SG2NAs in tissues is also regulated by differential polyadenylation and mRNA/protein stability. Further, the longer UTR is seen only in the brain mRNA from 1month old mouse and 8-10day old chick embryo. Like alternative splicing, differential polyadenylation of Sg2na transcripts is also conserved in evolution. Taken together, these results suggest a highly versatile and dynamic mode of regulation of SG2NA with potential implications in tissue development.

Secretory and transmembrane proteins undergo post-translational modifications and folding in the subcellular organelle, that is, endoplasmic reticulum (ER) to become functionally active. Various factors such as high oxidative stress, low glucose, calcium imbalance, and viral infections interfere with the ER protein folding functions, leading to accumulation of unfolded and misfolded proteins that activate downstream signal transduction pathways, termed as unfolded protein response (UPR). This UPR signaling is adaptive and restored the normal function of cells by decreasing protein synthesis, increasing the folding capacity of ER and degradation of misfolded proteins. If the stress condition is overwhelmed, then UPR signaling shifts to apoptotic pathways. However, cancer cells utilized these UPR signaling for their survival and progression as an adaptive mechanism. In this review, the authors discuss about the overview of ER stress and subsequent UPR signaling and various aspects of cancer as survival, proliferation, and angiogenesis in relation to UPR. Understanding the UPR signaling in relation to cancer will be further helpful in designing therapeutics against cancer.

SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.